High-throughput cell optoporation system based on Au nanoparticle layers mediated by resonant irradiation for precise and controllable gene delivery

The development of approaches based on genetically modified cells is accompanied by a constant intensive search for new effective and safe delivery systems and the study of existing ones. Recently, we developed a new plasmonic nanoparticle layers-mediated optoporation system that can be proposed for precisely controlled, high-performance laser transfection compatible with broad types of cells and delivered objects of interest. The main goal of the present study is to demonstrate the broad possibilities and advantages of our system for optoporation of several mammalian cells, classified as "easy-to-transfect" cells, namely HeLa and CHO lines, and "hard-to-transfect" cells, namely A431 and RAW 264.7 cells. We show the efficient delivery of various sized cargo molecules: from small molecular dyes propidium iodide (PI) with molecular mass 700 Da, control plasmids (3–10 kb) to fluorophore-labeled dextranes with masses ranging from 10 kDa up to 100 kDa. The performance of optoporation was investigated for two types of laser sources, 800-nm continuous-wave laser, and 1064-nm ns pulsed laser. We provided a comparative study between our system and commercial agent Lipofectamine for transient transfection and stable transfection of HeLa cells with plasmids encoding fluorescent proteins. The quantitative data analysis using flow cytometry, Alamar blue viability assay, and direct fluorescence microscopy revealed higher optoporation efficacy for hard-to-transfect A431 cells and Raw 264.7 cells than lipofection efficacy. Finally, we demonstrated the optoporation performance at the single-cell level by successful delivering PI to the individual CHO cells with revealed high viability for at least 72 h post-irradiation.

Section S1.Reagents Section S2.Plasmid DNA characteristics Section S3.UV-vis characterization of AuNS colloid and layer Section S4.Selection of optimal optoporation regimes for pulsed laser Section S5.Various types of biomolecules delivered to the HeLa cells using the optoporation system Section S6.Production of plasmid DNA preparations by molecular cloning Section S7.Obtaining a HeLa cell line with stable expression of the fluorescent protein gene Section S8.Optoporation of "hard-to-transfect" Raw 264.7 cells Section S9.Optotransfection vs lipofection cross-validation study results

Section S2. Plasmid DNA characteristics
We used commercial vectors based on the plasmid DNA carrying genes for fluorescent proteins with emission in a wide spectral range, expressed in the mammalian cells under the control of the CMV promoter.All vectors are equipped with cassettes with antibiotic resistance genes (neomycin) for selective selection in eukaryotic cells.In addition, they contain an additional SV40 promoter and specific antibiotic resistance cassettes (see below) for cloning and selection in prokaryotic cells.The number of copies in bacterial cells is 10-50 copies/cell.Below are the individual characteristics of the vectors (Table S1).

Section S6. Production of plasmid DNA preparations by molecular cloning Bacterial cultures
We used an endonuclease (endA) defective Escherichia coli XL1-Blue (no.632) strain, which was purchased from the collection of rhizospheric microorganisms of the IBPPM RAS.
Cultivation of E. coli bacteria, preparation of competent cells, and transformation with DNA plasmids (according to the heat shock mechanism) were carried out according to the generally available methods.[1].The selection of transformants and further maintenance of clones was carried out on a solid selective medium LB (Lisogenic Broth) containing 100 μg/ml of the antibiotic.Then, the selected colonies were subcultured onto a liquid selective medium, and plasmid DNA (pDNA) was extracted with the GeneJET Plasmid Midiprep Kit (Thermo Scientifics, USA) according to the manufacturer's recommendations.The isolated pDNA was either used immediately or stored at -20°C.The amount of highly purified plasmid preparation necessary for the experiments was obtained using the standard cloning technique [2] in E. coli XL1-Blue cells, shown in Fig. S4.The obtained transformants containing pDNA were cultured in a liquid nutrient medium, followed by extraction of pDNA in preparative amounts with a commercial plasmid isolation kit, then E. coli cells and pDNA preparations were cryopreserved for further use.As a result, pDNA preparations were developed: pGLuc, 650 µg; pGFP, 170 μg; pRFP -150 µg, purity and quantity were assessed by UV spectrophotometry at the 230/260/280 nm wavelengths.Restriction analysis (Fig. S4-p.7A) and control transfection of HeLa cells via lipofection (Fig. S4-p.8)showed complete agreement between the optical and molecular parameters of the original pDNA samples.

Characterization of plasmid preparations
The plasmid profile was obtained via restriction analysis using commercial enzymes EcoR1 and BamH1 according to the manufacturer's instructions.Briefly, 1 μg of pDNA and 1 μl of restriction enzyme in a supplied buffer were added to the reaction mixture, and the total volume of the mixture was adjusted to 25 μl with Milli-Q water.The reaction tubes were incubated for 5 h at 37°C, then the reaction was inhibited by adding 4 µl of glycerol-containing gel loading buffer to each tube.Restriction products were subjected to electrophoretic separation on a 1% agarose gel prepared in 1X TBE buffer (54 g Tris, 27.5 g boric acid, 146 g EDTA in 1 L distilled water) supplemented with 0.5 μg/ml EtBr.Electrophoresis was carried out for 40-50 min at a field strength of 6 W/cm.Optoporation of RAW 264.7 cells, which are monocyte-macrophage-like cells, was performed under the following pulsed laser irradiation modes: pulsed pulse energy 1.4 μJ, pulse duration 200 ns, pulse frequency 10 kHz, scanning speed 25 mm/s.For these cells, the minimum starting monolayer confluence of 50% was found.PI were used as a delivery agent (Fig. S6).Due to the possibility of precise tuning of the irradiation regimes, the PI was successfully delivered to the RAW 264.7 cells, within the high level of the maintained viability above 90% assayed by Alamar-test.

Section S9. Optotransfection vs lipofection cross-validation study results
Summarizing all the obtained data of transient transfection with control pDNA, a summary table S2 is presented below, clearly demonstrating the competitive advantages of the developed original technology of plasmonic optoporation in comparison with commercial lipocation agents.

Figure S1 -Figure S2 -
Figure S1 -Normalized extinction spectra of the original freshly synthesized AuNS colloid and the obtained 2-D layers on the surface of the 24-well plate bottom.

Figure S4 -
Figure S4 -Step-by-step scheme for the production and purification of pDNA by molecular cloning (1-5) in E. coli XL1-Blue cells; restriction analysis (7A) and phase-contrast and fluorescence microimages of HeLa cells, acquired 72 h post control transfection with LF (8).The scale bars correspond to 50 µm.

Section S7 .Figure S5 -
Figure S5 -Fluorescent images and phase-contrast images of HeLa-mCherry + optoporated cells (a) and lipofected (b) cells.The images were acquired at the 21th day after transfection.The scale bars correspond to 50 µm.

Figure S6 -
Figure S6 -Optoporation of RAW 264.7 cells.(a) fluorescent images and (b) phase contrast images of the cells; yellow and blue lines show the trajectory of the pulsed laser.The scale bars correspond to 50 µm.

Table S1 -
Plasmid DNA used in the work.

Table S2 -
Cross validation study results of optotransfection vs lipofection for transient transfection of several types cell lines.